Glucagon-like peptide-1 (hereinafter referred to as GLP-1) functions to induce various biological effects, including the stimulation of insulin secretion, the suppression of glucagon secretion, the promotion of satiety, the inhibition of gastric or intestinal motility, the augmentation of glucose use, and the induction of weight loss. Also, GLP-1 is known to have functions of preventing the degeneration of pancreatic cells caused by the progression of type II diabetes, that is, non-insulin dependent diabetes mellitus (NIDDM), and of promoting the growth of nascent-cells so as to recover insulin secretion. Particularly, the conspicuous feature of GLP-1 resides in the ability to stimulate insulin secretion without the concomitant induction of hypoglycemia, which is a major risk of insulin therapy or oral therapy for inducing an increase in insulin expression. In addition, none of the adverse events associated with the long-term administration of the hypoglycemic agent sulfonylurea, such as the apoptosis and necrosis of pancreatic β-cells, are found with GLP-1. Therefore, GLP-1 is regarded as very useful in the treatment of type II diabetes.
However, therapy with GLP-1 is restricted in utility not only because the activity of GLP-1 itself is insufficient, but also because two truncated, native GLP-1 molecules, GLP-1 (7-37)OH and GLP-1(7-36)NH2, have very short plasma half lives. In detail, GLP-1 is one of the substrates of endogenous dipeptidyl peptidase-IV, being inactivated by the removal of the N-terminal histidine-alanine dipeptide moiety (aa 7 and 8), which is known to be a major cause of the short biological life span thereof (O'Harte et al., 2000).
There are many approaches to reduce the degradation of GLP-1 or to extend the plasma lifespan of GLP-1 while maintaining the biological activity thereof, including the use of DPP-IV inhibitors (P93/01, NVP-LAF237, NVP-DPP728, 815541A, 823093, MK-0431, etc.), and the use of ligands acting to GLP-1 receptors or GLP-1 derivatives (exendin, liraglutide, GLP-1/CJC-1131, etc.).
Exendins, first discovered by John Eng (U.S. Pat. No. 5,424,286), are a family of polypeptides capable of reducing the blood level of glucose. Exendin-4 has the following amino acid sequence, sharing partial homology (53%) with GLP-1(7-36)NH2 (Goke et al., 1993).
His1-Gly-Glu-Gly-The-Phe-The-Ser-Asp-Leu-Ser- Lys12-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe- Ile-Glu-Trp-Leu-Lys27-Asn-Gly-Gly-Pro-Ser-Ser- Gly-Ala-Pro-Pro-Pro-Ser-NH2
Exendins are found in the venom of the beaded lizard and the gila monster: exendin-3 is extracted from the poison of the beaded lizard, Heloderma horridum and exendin-4 is present in the gila monster, Heloderma suspectum. Exendin-4 is different from exendin-3 only in amino acid position 2 and 3. In mammals, exendins are resistant to DPP-IV digestion so that their half life is longer than that of GLP-1, which is 2 min or less (Kieffer T J et al., 1995). In an in vivo test, exendins were found to have a half life of 2˜4 hrs and reach a sufficient level in the blood upon two or three abdominal administrations per day (Fineman M S et al., 2003). In addition, it is known that exendin-4 functions to regulate gastrointestinal motility, decrease food intake and inhibit blood glucagon (U.S. Pat. Nos. 6,858,576, 6,956,026 and 6,872,700). With regard to the effect of exendin-4 on glycemic control, HbA1c levels, the amounts of hemoglobin bound to glucose in blood, were measured to decrease by 1% or less in both groups administered with exendin-4 alone and in combination with an anti-diabetic agent, such as sulfonylurea or metformin (Egan J M et al., 2003). Recently, the sale of synthetic exendin-4 under the brand name of Byetta has been approved by the FDA.
Polyethylene glycol (PEG), a polymer having the chemical structure of HO—(—CH2CH2O—)n—H, is strongly hydrophilic, and thus it can increase the solubility of medicinal peptides when it is coupled therewith. PEG, when properly coupled with medicinal peptides, increases the molecular weight of the modified peptides to protect them from renal filtration, cells recognizing exogenous antigens, antibodies, and enzymatic degradation while their major biological functions, such as enzymatic activity and receptor binding are maintained. When its molecular weight falls within the range from 1,000 to 100,000, PEG can be properly coupled with peptides. PEG with a molecular weight of 1,000 or higher is known to have very low toxicity. PEG ranging in molecular weight from 1,000 to 6,000 is distributed throughout the body and metabolized in the kidneys. Particularly, PEG having about MW 40,000 is distributed in blood and the liver and the metabolism thereof is conducted in the liver.
Generally, when administered via parenteral routes, medicinally or pharmaceutically useful proteins may be antigenic in the body, for the most part, poor in water solubility and have a short retention time in the body. Approaches to overcome these shortcomings are under study. U.S. Pat. No. 4,179,337 teaches that when proteins or enzymes conjugated with PEG are used as therapeutics, they enjoy advantages from PEG, including a decrease in antigenicity and an increase in water solubility and in retention time. Since this patent was granted, many attempts have been made to couple biologically active proteins with polyethyleneglycol to overcome various shortcomings. For instance, ribonuclease and superoxide dismutase were coupled with PEG (Veronese et al., 1985) and when coupled with polymers including PEG, proteins were reported to show increased water solubility in U.S. Pat. Nos. 4,766,106 and 4,917,888. In addition, it was disclosed that recombinant proteins coupled with PEG or other polymers are decreased in antigenicity and increased in retention time in U.S. Pat. No. 4,902,502.
Despite such advantages, PEG limits the function of the proteins conjugated therewith. In detail, PEG is conjugated with a protein via a covalent bond to a lysine residue(s) of the protein. When the lysine residue is directly responsible for the activity of the protein, the protein conjugated with PEG cannot perform the biological function any more. Furthermore, because the bonding of PEG with lysine residues takes place randomly, many kinds of PEG-protein conjugates may result, making it complicated to separate the desired ones from the conjugate mixture.
There have been many attempts to conjugate GLP-1 or analogues thereof and exendin, which are therapeutically useful and have a short half life with PEG. PCT Publication No. 04/093823 reports that, when coupled with one or more polyethyleneglycol molecules, GLP-1 compounds or derivatives thereof were increased in half life and underwent degradation at low rates. The incorporation of one or two cysteine residues into specific amino acid residues of a peptide of interest provides one or two thiol groups, which then act as sites with which PEG or derivatives thereof (particularly, PEG-maleimide) can form a covalent bond so as to produce polyethylene-glycolated GLP-1 compounds. In an alternative, GLP-1, analogues or fragments thereof are covalently bonded to polyethylene glycol or polyethylene glycol derivatives at lysine residues or the carboxyl terminus to produce modified molecules which are extended in half life and decreased in degradation rate. In PCT Publication No. 04/093823, it was disclosed that PEG or derivatives thereof. Can form covalent bonds with cysteine resides at positions 26 and 34, lysine resides at positions 18, 22 and 26, or the carboxyl terminal residue of GLP-1 or analogues. It is also suggested that one peptide molecule may be conjugated with one to six PEG molecules, which preferably range in molecular weight from 20,000 to 40,000 daltons. The polyethylene-glycolated GLP-1 compounds prepared according to the method of PCT Publication No. 04/093823 were disclosed to have longer half lives and lower degradation rates in relation to natural GLP-1 or Val8-GLP-1(7-37)OH, while retaining all or some of the biological activity of natural GLP-1.
U.S. Pat. No. 6,924,264 addresses novel modified exendin and exendin agonist analogs. The modified exendin-4 has three amino acid residues (N-terminal histidine, lysine 12 and 27) capable of being linked to PEG. The PEG used in U.S. Pat. No. 6,924,264 has a molecular weight ranging from 5000 to 12,000 daltons and can form covalent bonds with epsilon-amino groups of two lysine residues. In contrast to natural GLP-1, which undergoes primary proteolysis, the exendins increased in molecular weight are removed from plasma through renal filtration.
As described above, various approaches can produce exendins which are increased in lifespan and improved in efficiency in comparison with natural exendins. However, when two or more PEG molecules are covalently bonded to biologically active pepti des, such as GLP-1 or exendins, the resulting conjugates may show disadvantageous properties insufficient to be used as drug for example, may be decreased in stability and biological activity.
Leading to the present invention, the intensive and thorough research into an improvement in the pharmaceutical effect of exendin, conducted by the present inventors, resulted in the finding that the modification of exendin at a specific amino acid position with PEG or a derivative thereof produces PEG-exendin conjugates which are improved in retention time within the body without loss of the biological activity of natural exendin, and have exellent pharmacokinetic profiles and pharmaceutical effects, thereby decreasing in dose or administration number in comparison with natural exendin.